Amyloid-β Causes NMDA Receptor Dysfunction and Dendritic Spine Loss through mGluR1 and AKAP150-Anchored Calcineurin Signaling

Abstract

Neuronal excitatory synapses are primarily located on small dendritic protrusions called spines. During synaptic plasticity underlying learning and memory, Ca²⁺ influx through postsynaptic NMDA-type glutamate receptors (NMDARs) initiates signaling pathways that coordinate changes in dendritic spine structure and synaptic function. During long-term potentiation (LTP), high levels of NMDAR Ca²⁺ influx promote increases in both synaptic strength and dendritic spine size through activation of Ca²⁺-dependent protein kinases. In contrast, during long-term depression (LTD), low levels of NMDAR Ca²⁺ influx promote decreased synaptic strength and spine shrinkage and elimination through activation of the Ca²⁺-dependent protein phosphatase calcineurin (CaN), which is anchored at synapses via the scaffold protein A-kinase anchoring protein (AKAP)150. In Alzheimer's disease (AD), the pathological agent amyloid-β (Aβ) may impair learning and memory through biasing NMDAR Ca²⁺ signaling pathways toward LTD and spine elimination. By employing AKAP150 knock-in mice of both sexes with a mutation that disrupts CaN anchoring to AKAP150, we revealed that local, postsynaptic AKAP-CaN-LTD signaling was required for Aβ-mediated impairment of NMDAR synaptic Ca²⁺ influx, inhibition of LTP, and dendritic spine loss. Additionally, we found that Aβ acutely engages AKAP-CaN signaling through activation of G-protein-coupled metabotropic glutamate receptor 1 (mGluR1) leading to dephosphorylation of NMDAR GluN2B subunits, which decreases Ca²⁺ influx to favor LTD over LTP, and cofilin, which promotes F-actin severing to destabilize dendritic spines. These findings reveal a novel interplay between NMDAR and mGluR1 signaling that converges on AKAP-anchored CaN to coordinate dephosphorylation of postsynaptic substrates linked to multiple aspects of Aβ-mediated synaptic dysfunction.

Keywords: AKAP; NMDA receptor; amyloid-β; calcineurin; dendritic spine; mGluR1.

Figure 1.

AKAP150-anchored CaN is required for Aβo-mediated reduction in NMDAR Ca²⁺ signals. A, Time series showing a QCT visualized in a dendritic spine (red box) from a mouse hippocampal neuron transfected with GCaMP6s. The traces below show GCaMP6s signal from a single dendritic spine before (black) and after (red) APV addition. B, Representative effect of APV on QCT frequency in WT neurons (n = 93 spines from 1 neuron). C, Representative effect of APV on QCT frequency in AKAPΔPIX neurons (n = 36 from 3 neurons). D, Comparison of baseline QCT amplitudes from WT and AKAPΔPIX neurons (n = 577, from N = 17 independent cultures). E, Representative QCT traces measured from neurons cultured from WT animals from the same spine before and after a 45 min incubation with vehicle (left), 100 nM (middle), or 500 nM (right) Aβo. F, Compiled QCT data from WT neurons plotting the average change in amplitude 45 min post-Aβo treatment compared with baseline measurements prior to Aβo treatment. Vehicle, n = 104 spines, 8 neurons, N = 3; 100 nM, n = 104, 8 neurons, N = 3; 500 nM Aβo, n = 107, 9 neurons N = 3; frequency (n = 108, 8–9 neurons, N = 3 for all conditions). **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett's. G, GluN2B is the primary target of Aβo-mediated Ca²⁺ amplitude decrease. Data represented as within-single-spine change in QCT amplitude post 45 min Aβo treatment (left panel) or a 5 min, 5 μM ifenprodil treatment followed by addition of 500 nM Aβo for 45 min (right panel), showing no further QCT decrease (n = 26 spines from 1 neuron). ****p < 0.001 by one-way ANOVA with Tukey's. H, Same as E except measurements were made from AKAPΔPIX neurons. I, Same as F, except measurements were made from AKAPΔPIX neurons. Amplitude: vehicle, n = 104, 9 neurons, N = 3; 100 nM Aβo, n = 108, 9 neurons, N = 3; 500 nM Aβo, n = 108, 8 neurons, N = 3).

Figure 2.

Aβo application does not alter GluN2B Y1472 or S1480 phosphorylation. A, Levels of GluN2B Y1472 phosphorylation after 45 min Aβo application in WT (vehicle, 100 and 1,000 nM Aβo, N = 7; 500 nM Aβo, N = 6) and (B) AKAPΔPIX (vehicle and 500 nM Aβo, N = 4; 100 and 1,000 nM Aβo, N = 5) hippocampal mouse cultures. Top, Representative Western blot. Bottom, Quantification. C, Levels of GluN2B S1480 phosphorylation after 45 min Aβo application in WT (N = 4 for all conditions) and (D) AKAPΔPIX (N = 5 for all conditions) hippocampal mouse cultures.

Figure 3.

Aβo application results in a dose-dependent decrease in GluN2B S1166 phosphorylation mediated by AKAP-anchored CaN. A, Levels of GluN2B S1166 phosphorylation after 45 min Aβo application in WT (N = 7 for vehicle, 100 and 500 nM Aβo; N = 3 for 1,000 nM Aβo) and (B) AKAPΔPIX (vehicle, N = 5; 100 nM Aβo, N = 8; 500 nM Aβo, N = 7; 1,000 nM Aβo, N = 3). Left, Representative Western blot; right, quantification. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett's. C, Hippocampal neuronal lysates were prepared after incubation (45 min) in QCT imaging conditions (in mM: 130 NaCl, 5 KCl, 10 HEPES, 30 glucose, 2.5 CaCl₂, 0.003 glycine, and 0.002 TTX, pH 7.4) to assess protein phospho-states corresponding to baseline Ca²⁺ amplitudes in WT and AKAPΔPIX mouse cultures. Quantification of pS99 PKA RIIα levels normalized to total PKA RIIα (WT, N = 6; PIX, N = 6; ****p < 0.001 by t test). Note: the pS99 blot always contained a prominent smaller band below the phospho band that did not colocalize with the total PKA RIIα protein signal and was excluded from quantification of the phospho band). D, Quantification of pS1166 GluN2B levels normalized to total GluN2B (WT, N = 6; PIX, N = 6). Loading control is α-tubulin. D, Comparison of baseline QCT amplitudes between WT and PIX mouse cultures (WT, n = 577, N = 17 independent cultures; PIX, n = 607, N = 17 independent cultures, p > 0.05.).

Figure 4.

mGluR1 activation is required for Aβo-mediated deficits in NMDAR Ca²⁺ influx and alterations in GluN2B pS1166. A, Comparison of changes in QCT amplitudes in WT DIV14 neurons treated for 45 min with vehicle (black bar), 500 nM Aβo (red bar), 25 μM MTEP alone (blue bar) or 500 nM Aβo plus MTEP (purple bar). Note MTEP was added 15 min prior to Aβo addition. (Vehicle, n = 108 spines; Aβo, n = 104 spines; MTEP, n = 104 spines, MTEP + Aβo, n = 107 spines; N = 3 independent cultures). *p < 0.05, ****p < 0.0001 by one-way ANOVA with Tukey's. B, Comparison of changes in QCT amplitude of WT DIV14 neurons treated for 45 min with vehicle (black bar), 500 nM Aβo (red bar), 30 μM LY367385 alone (blue bar), or 500 nM Aβo plus LY367385 (purple bar). Note LY367385 was added 15 min prior to Aβo addition. (Vehicle, n = 95; Aβo, n = 97; LY367385, n = 105; LY367385 + Aβo, n = 98; N = 3 independent cultures). *p < 0.05 by one-way ANOVA with Tukey's. C, Levels of pS1166 GluN2B, total GluN2B, with α-tubulin as a loading control. Lysates collected after treatment in the same corresponding conditions as in A and B. (Vehicle, N = 8; 500 nM Aβo, N = 8; LY367385, N = 7; LY367385 + Aβo, N = 8; MTEP, N = 5; MTEP + Aβo, N = 5.) *p < 0.05, ***p < 0.001 by one-way ANOVA with Dunnett's.

Figure 5.

Disruption of AKAP–CaN anchoring protects hippocampal CA1 LTP from inhibition by Aβo. A, Acute WT hippocampal slices (2–3-week-old) were either treated with 100 nM Aβo (red) or vehicle (black) for 30 min prior to LTP induction at t = 0 (vehicle, N = 7; Aβo, N = 5). Representative EPSP traces recorded from vehicle-treated and Aβo-treated WT slices pre- and 55 min post-LTP induction are shown below. B, The effect of 30 min pre-incubation with 100 nM Aβo on LTP in AKAPΔPIX mice (N = 6 for both conditions). Representative EPSP traces recorded from vehicle-treated and Aβo-treated AKAPΔPIX slices are shown below. **p < 0.01 by two-way ANOVA.

Figure 6.

AKAP–CaN is required for Aβ-mediated dendritic spine loss and activation of the F-actin severing protein cofilin. A, Spine density following 24 h of exposure to vehicle or varying Aβo concentrations was quantified in 14–16 DIV hippocampal neurons cultured from WT mice (left, representative images; right, quantification; control, n = 31 cells, N = 3 independent cultures; 100 nM Aβo, n = 32, N = 3; 500 nM Aβo, n = 34, N = 3; 1 μM Aβo, n = 27, N = 4). B, Spine density following 24 h of exposure to vehicle or varying Aβo concentrations was quantified from AKAPΔPIX mouse hippocampal neurons (left, representative images; right, quantification; control, n = 36, N = 4; 100 nM Aβo, n = 35, N = 4; 500 nM Aβo n = 38, N = 4; 1 μM Aβo, n = 16, N = 4). ***p < 0.001 or ****p < 0.0001 by one-way ANOVA with Dunnett's. C, Levels of phosphorylated (inactive) cofilin after Aβo treatment in WT and (D) AKAPΔPIX mouse neurons (left, representative images; right, quantification; N = 5 for all conditions). *p < 0.05 or **p < 0.01 by one-way ANOVA with Dunnett's. Scale bar, 10 μm.

Figure 7.

mGluR1 activation is critical for Aβo-mediated dendritic spine loss. A, Top, Representative dendrites of WT neurons pretreated with vehicle, 30 μM LY367385, or 25 μM MTEP for 15 min, followed by a 24 h vehicle or Aβo application in the continued presence of inhibitors. Bottom, Quantification of spines/10 μm dendrite (WT, n = 27 cells; Aβo, n = 27; LY367385, n = 26; LY367385 + Aβo, n = 27; MTEP, n = 27, MTEP + Aβo, n = 27; N = 3 independent cultures). LY, LY367385. B, Levels of phospho-cofilin, cofilin, and α-tubulin after treatments described in A. N = 6 independent cultures. *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Tukey's.

Figure 8.

Model for mGluR1 and AKAP150-anchored CaN signaling in Aβ regulation of NMDAR Ca²⁺ influx and F-actin dynamics in dendritic spines. AKAP–CaN regulation of PKA RIIα, GluN2B, and cofilin phosphorylation under basal conditions of homeostasis (left) and in response to acute application of Aβo (right). Created with BioRender.com.